The regulation of major histocompatibility complex (MHC) class II genes is central to a healthy and active immune system. Discerning the molecular mechanism(s) underlying their regulation is therefore important for our ability to understand, design, and provide immune based therapies for infection, autoimmune disease, and organ transplantation. MHC class II genes are regulated by four transcription factors whose genes are deficient in a rare, genetic-based immunodeficiency termed the bare lymphocyte syndrome (BLS). Three of the "BLS factors" (RFX-B, RFX5, and RFXAP) are subunits of RFX, a factor that binds to all MHC class II promoters and interacts directly with CIITA, the fourth BLS factor. All four proteins are required for MHC class II expression. With the genes for all of the factors cloned, the opportunity now exists to characterize the function of these important transcription factors and the gene system they control. Critical questions about the properties and function of the RFX subunits and CIITA remain and will be addressed in the aims of this proposal. 1) RFX5 and RFXAP but not RFX-B are functionally imported into the nucleus, and RFXAP appears to have a potent export signal, yet the regions of these proteins signaling these processes have not been defined. It is also not known how RFX-B enters the nucleus, what role RFXAP export plays, and how these processes are regulated. KPNA7, a factor involved in nuclear import, was found to be regulated by CIITA. Does this factor function in MHC expression? 2) The phosphorylation of the three RFX subunits was shown to correlate with MHC class II activity, yet the sites of phosphorylation and their exact role is unknown. Does phosphorylation affect import? Is phosphorylation necessary for RFX interactions with CIITA? 3) Finally, CIITA is transcribed in a cell type dependent manner from four promoters, each providing a distinct amino terminus. The promoter I (PI) isoform, which is expressed in dendritic cells and macrophages, is considerably more potent in its ability to activate transcription due to a caspase recruitment domain not found on the other isoforms. The importance and functional role of this isoform in the development of immune responses is unknown and will be examined using knock-in mice expressing the weaker isoforms of promoter III and IV isoforms driven from promoter I. Thus, the central goal of this application is to understand fully the mechanisms by which these transcription function to regulate this important gene system. The results of these studies will ultimately identify targets for immune based therapies for the diseases listed above. [unreadable] [unreadable] [unreadable] [unreadable]